Neisseria meningitidis has the typical envelope of Gram-negative bacteria, consisting of a cytoplasmic membrane, a peptidoglycan layer and an outer membrane with three layers, which, together with the capsular polysaccharide, constitute the bacterial wall.
From an immunological point of view, those structures which may interact with the immune system in any of the stages of the microorganism-host relationship and especially the first stages, i.e., the most external components, are of greater importance. Classification of this microorganism has been made on the basis of groups, types and subtypes, through techniques utilizing polyclonal (Frasch, C. E. and Chapman, 1973, J. Infect. Dis. 127: 149-154) or monoclonal antibodies (Hussein, A., Monoclonal Antibodies and N. meningitidis. Proefschrift. Utrecht, Nederland, 1988).
Nowadays 12 serogroups are known: A, B, C, X, Y, Z, 29-E, W-135, H, I, K, and L (Ashton, F. E. et al., 1938, J. Clin. Microbiol. 17: 722-727; Branham, S. E., 1956, Can. J. Microbiol. 2: 175-188; Evans, A. C., 1920, Lab. Bull. 1245: 43-87; Shao-Qing, et al., 1972, J. Biol. Stand. 9: 307-315; Slaterus, K. W., 1961, Ant. v. Leeuwenhoek J. Microbiol. Serol. 29: 265-271). Such groups have been differentiated according to the capsular polysaccharide (CPS) which vary among each of these groups in certain antigenic determinants (Branham, S. E.,1940, Bacteriol Rev. 4: 59-96; Frasch, C. E. et al, 1985, Rev. Infect. Dis. 7: 504-510).
In spite of variations in epidemiology in different regions of the world and a lack of adequate systems of sanitary control in some countries, it has been possible to establish that, among all these groups, at present only groups A, B, C, Y, and W-135 are responsible for more than 90% of the meningitidis cases in the world (Abdillahi, H., 1988, Proefschrift, pg. 13 Utrecht, Nederland). Other authors ascribe more than 95% (Frasch, C. E. Eur. J. Microbiol. 4: 533-536) of cases to these five groups.
The capsular polysaccharides, purified and characterized, and used so far as vaccines have proved efficient enough to control the outbreaks and epidemics of the groups A, C, Y, and W-135, as mono-, di, tri, or tetravalent vaccines (Gold et al., 1969-1970, Bull. WHO 45: 272-282; Gotschlich et al., 1969, J. Exp. Meal. 129: 134-136; Hankins, 1982, Proc. Soc. Biol. Med. 169: 54-57). In spite of deficiencies still to be solved such as: poor or no-response to polysaccharide C in children under 2 years of age; thermalability of polysaccharide A; difficulties regarding the induction of tolerance after re-vaccination with polysaccharides A and C, to the point that in some places increase of meningitidis cases has been detected (Marzochi, K. B. F. Meningitidis Meningococcica. Tese (doutorado) Universidade Federal do Rio de Janeiro, Brazil (328 pg.), 1985). Now the polysaccharide vaccines are an effective enough weapon in the struggle against disease caused by groups A, C, Y, and W-135, but not against group B, the capsular polysaccharide of which is a very poor immunogen (Wyle et al., 1972, J. Infect. Dis. 126, 514-522; Zollinger, et al., 1979, J. Clin. Invest. 63: 836-834; Jennings et al., 1981, J. Immunol. 127: 104-108). It may be due to different causes such as the cross reactivity with antigens of the host itself (brain sialoglycopeptides), conformational changes suffered by these polysaccharides when they are purified, and their great sensitivity to enzymatic and other types of degradation occurring in the organism, such as: rapid esterification, etc. (Lifely, M. R. and Moreno, C., 1986, Lancet, Jan. 25, 214-215). The final result is that, despite the intense work of important researchers and companies such as, the Wellcome Foundation, it has been impossible until now to produce from the group B polysaccharide a satisfactory immunogen. Because of these reasons, the general methods for vaccines from the other polysaccharide groups used for vaccines do not work for group B. Because serogroup B is today the main cause of the meningococcal disease in most temperate counties, as well as other regions throughout the world, it is desirable to obtain alternative vaccines. Every attempt at finding these alternative vaccines should therefore be based upon non-capsular antigens, especially those in the outer membrane, taking into account their significance in the microorganism-host relationship.
Almost every research group has focused its attention on the proteins, since the other relevant components would be the lipopolysaccharides (LPS), but their highly toxic nature, their high pyrogenic activity and their mitogenic capacity have prevented considering this element as a vaccine option, although it is the most important in the pathogenesis of the meningococcal disease.
The principal proteins in the outer membrane, both from groups B and C give rise to subdivisions of these groups in serotypes (from now on we shall only refer to group B). They are based on the immunologic specificity of these principal proteins detected in reactions with polyclonal and monoclonal antibodies in different techniques (Abdillahi, H. Proefschrift, Utrecht, Nederland, 1988). Group B has already been subdivided into more than 18 serotypes.
Preferred techniques include double immunodiffusion, counterimmunoelectrophoresis, radioimmunoassay, ELISA, SPRIA, coagglutination using protein A coated Staphylococcus aureus to fix monoclonal antibodies or other solid carriers, agglutination by latex, etc. (Gold, R. and Wyle, F. A., 1970, 1: 479-484; Jones, O. M. and Tobin, B. M., 1976, J. Clin. Pathol.29: 746-748; Zollinger, W. D. and Mandrell, R. E., 1977, Infect. Immun. 5: 98-102; Frasch, C. E. and Chapman, S. S., 1973, Infect. Dis. 127: 149-154; Danielsson, D. and Olsen, 1979, J. Clin. Pathol. 32: 136-142).
With the aid of monoclonal antibodies it has even been possible to subdivide further different serotypes correlating them with their degree of "virulence", e.g. serotype 2 was subdivided into 2a, 2b and 2c (Abdillahi, H. and Poolman, J. T.,1988, Proefschrift. Ch. 6 pg. 69-78, Utrecht, Nederland; Poolman, J. T. et al., 1980, J. Gen. Microbiol. 116: 465-473).
It is obvious that, by using the principal proteins of the outer membrane as a basis for the immunogen, we are preparing a specific serotype immunogen. In spite of the fact that it has been found that among all the serotypes only a few are disease agents (Frasch, C. E., et al, 1972, Seminars in Infectious Diseases, Vol. 1, S.I.M. Book Corp., N.Y. 304-337) while others seldom are, it would be impossible to prepare an immunogen based on a serotype, which would have sufficient antigenic determinants for all the serotypes causing disease. Therefore, serotype vaccines as such have a small range of effectiveness, aiming only at the specific serotype with which the vaccine was elaborated. This has been the history of almost all the protein vaccines prepared in the last few years and that has been among several others, one of their major limitations. Vaccines based on serotype 2a proteins have been prepared from vesicles of the outer membrane (Frasch, C. E. et al., 1982, Infect. Immun. 37: 271-280) and certain variants of those vaccines have been studied regarding their immunogenicity and toxicity in animals (Peppler et al., 1982, Infect. Immun. 37: 264-270) and assessed for their safety and immunogenicity in voluntary adults (Frasch, C. E. et al., 1982, Sem. Infect. Dis., 4; Zollinger et al, 1979, J. Clin. Invest. 63: 836-848).
Other research work also reveals the importance of the solubility of these vaccines (Frasch, C. E. et al., 1982, Sem. Infect. Dis., 4; Frasch, C. E., 1978, J. Exp. Med. 147: 629-644).
The benefits of the use of capsular polysaccharides and different adjuvants of aluminum hydroxide or phosphate have also been reported by numerous authors (Frasch, C. E., 1983, Med. Microbiol. Vol. 2 Acad. Press, N.Y.). In some cases a careful study has been carried out on the correlations among the proportions of those components, their electron microscopy picture and their response in different animals, including primates and humans of different age groups (Campa, C. et al, 1986).
In our research work we even found the solution for the lack of immunogenicity in children under 2 years of age.